摘要
目的:利用SHN3免疫特异肽段亲和层析纯化抗SHN3多克隆抗体。方法:将制备多克隆抗体SHN3免疫原片段(99bp)划分为4段互相重叠等长的基因片段(48bp),构建原核表达重组质粒并转化E.coil的BL21(DE3)菌株。IPGT诱导重组蛋白表达,Western blotting筛选并人工合成与抗体免疫结合特异性片段,亲和层析纯化抗体,Western blotting检测纯化结果。结果:成功地筛选到SHN3免疫原特异肽段part-3,以亲和层析的方式纯化多克隆抗体,纯化后免疫特异性提高。结论:亲和层析的方式纯化得到的SHN3多克隆抗体抗体,有效地提高免疫特异性,为后续SHN3蛋白功能研究和应用奠定了基础。
Objective:To purify polyclonal antibodies against SHN3 by affinity chromatography with immunologic specificity peptide frag- ment. Method:SHN3 immunologic specificity gene (90bp)was divided 4 equal length parts(48bp). And subcloned into prokaryotic ex- pression vector Pmal - C2. These recombinant vectors were transformed into E. coliBL21 ( DE3 ). Recombinant peptide expression were in- duced by IPTG. Western blotting method was used to screen the best immunologic specificity peptide fragment. The polyclonal antibodies was purified by affinity chromatography and checked by Western blotting. Result:The immunologic specificity peptide fragment part -3 of SHN3 was screened successfully, and the SHN3 polyclonal antibodies were purified with this peptide by affinity chromatography. The affini- ty chromatography of SHN3 polyclonal antibodies was improved significantly. Conclusion: Affinity chromatograph for SHN3 polyclonal anti- bodies purification was successfully established and immunologic specificity was significantly improved. The method laid a foundation for subsequent SHN3 research and application.
出处
《生物技术》
CAS
CSCD
北大核心
2014年第1期51-54,共4页
Biotechnology
基金
国家自然科学基金项目("Dcf1基因对大脑发育神经元迁移及树突棘形成机制的研究"
No.81271253
"Dcf1基因在大脑发育过程的作用机制"
No.31070954)资助
关键词
抗体纯化
原核表达
亲和层析
免疫特异性
Antibody purification
Prokaryotic expression
Affinity chromatography
Immunologic specificity